Luminescence method for luciferin/luciferase system and luminescent reagent

ABSTRACT

A luminescence method for a luciferin/luciferase system which comprises reacting a luciferin solution containing an increased amount of dissolved carbon dioxide with luciferase; and a luciferase assay reagent comprising a luciferin solution containing an increased amount of dissolved carbon dioxide. Thus, a method of luminescent assay excellent in continuity and stability is provided as a substitute for the conventional luminescent luciferase assay featured by rapidity and high sensitivity.

TECHNICAL FIELDS

The present invention relates to a luminescence method forluciferin/luciferase system, which stabilizes a luminescent reaction anda luciferase assay reagent. The luminescence method forluciferin/luciferase system and the luciferase assay reagent, providedby the present invention, can be applied to all assay systems using aluciferase enzyme as a reporter and signal.

BACKGROUND

In a luminescent reaction with luciferin/luciferase in vitro,conventionally, the luminescence pattern thereof is observed in a flashstate. Therefore, it is impossible to measure the luminescent reactionaccurately without using a device having a specific mechanism ofinjecting a reagent. However, K. V. Wood et al. has established aluminescence system having a relatively long half-life period ofluminescence as long as 5 minutes. (Wood, K. V. Recent advantage andprospects for beetle luciferase as genetic reporters. In:Bioluminescence and Chemiluminescence current status. Proceeding of theVIth International Symposium on Bioluminescence and Chemiluminescence,Cambridge, September 1990. P.543. Ed. by P. Stanley and L. J. Kricka.).Owing to this, it has become possible to accurately measure aluminescence unit in a luciferin/luciferase system with a luminometer ora liquid scintillation counter which does not have an automaticreagent-injecting device. So, a reporter assay method wherein luciferasegenerated as a reporter in a culture cell is measured at a highsensitivity has been widely used. However, when the reporter assaymethod is used in a High Throughput screening for developing amedication, the luminescence half-life period of 5 minutes isinsufficient. For example, for measuring many samples by using 96 wells,luminescent reagents must be added several times and measurement must becarried out each time. Like this, restrictions on operation andmeasurement exist.

Further, luciferin contained as a luminescent substrate in a luminescentreagent is apt to be oxidized in a solution during storage, to form anoxyluciferine. It is well-known that the oxyluciferin inhibits theenzyme reaction of a luciferase. Conventional luminescent reagentscontaining luciferin as a luminescent substrate have a big problem thata luminescent activity deteriorates due to the oxidation of theluciferin. This problem prevents the further expansion of uses for theassay using luciferase as a reporter. Therefore, the development of anew stabilization method is expected.

It is an object of the present invention to provide a luminescencemethod for luciferin/luciferase system, which extends the half-lifeperiod of a luminescent reaction, and a luciferase assay reagent.

It is an another object of the present invention to a luminescencemethod for luciferin/luciferase system, which prevents the deteriorationof a luminescent activity and gives a stable luminescence reaction, anda Luciferase assay reagent.

DISCLOSURE OF THE INVENTION

According to the present invention, there is provided a luminescencemethod for luciferin/luciferase system, which comprises reactingluciferin solution containing an increased amount of dissolved carbondioxide with luciferase.

According to the present invention, further, there is provided aluminescence method for luciferin/luciferase system, wherein the pH ofthe luciferin solution is adjusted in the range of from 5.0 to 8.5.

According to the present invention, further, there is provided aluminescence method for luciferin/luciferase system, wherein the amountof the dissolved carbon dioxide in the luciferin solution is increasedby adding a dry ice.

According to the present invention, further, there is provided aluminescence method for luciferin/luciferase system, wherein the amountof the dissolved carbon dioxide in the luciferin solution is increasedby introducing a carbon dioxide gas.

According to the present invention, further, there is provided aluciferase assay reagent which comprises a luciferin solution containingan increased amount of dissolved carbon dioxide.

According to the present invention, further, there is provided aluciferase assay reagent which comprises a first component containing adried luciferin and a second component of a solution containing anincreased amount of dissolved carbon dioxide.

PREFERRED EMBODIMENT OF THE INVENTION

In the present invention, the term "luciferase" refers to allluciferases concerning a bioluminescence, such as a firefly.luciferase,a renilla.luciferase and a cypridina hilgendorfii.luciferase.

In the present invention, the term "luciferin" refers to luminescentsubstrates corresponding to luciferases concerning a bioluminescence,such as a firefly.luciferase, a renilla.luciferase and a cypridinahilgendorfii.luciferase.

As a method of increasing the amount of dissolved carbon dioxide in aluciferin solution as a luminescent reagent for a luciferase in thepresent invention, a method of adding a carbon dioxide gas or a dry iceis preferred. Examples of the method include even a method wherein aninorganic compound, such as sodium bicarbonate, which generates a gas byadding it into water, is added. However, it is preferred to use a carbondioxide gas or a dry ice which scarcely impairs living things such asmicroorganism and which does not generate a chemical reaction or thelike.

Owing to the use of a carbon dioxide gas, the luminescence reaction ofluciferase is continued for a long time. Concurrently, a luminescencesubstrate, luciferin, in the solution can be protected from oxidationdeterioration due to dissolved oxygen.

For controlling the pH of a luminescence solution, in the presentinvention, an inorganic acid such as hydrochloric acid, sulfuric acid,phosphoric acid and boric acid, an organic acid such as acetic acid,tartaric acid, malic acid, propionic acid, mucic acid, benzoic acid,maleic acid, succinic acid or fumaric acid, an inorganic alkalinecompound such as sodium hydroxide, potassium hydroxide, aqueous ammonia,sodium carbonate and potassium carbonate, or an organic base such asaniline, pyridine, imidazole, monomethylamine, dimethylamine,trimethylamine, monoethylamine, diethylamine, triethylamine, ethanolamine, diethanol amine or triethanol amine, is added. By controlling apH at a preferable pH value of pH 5 to 8.5, the shift to an alkalineside is prevented during a luminescence reaction, which can serve tomaintain the enzyme activity of luciferase.

The luminescence reaction is extremely prevented when the pH value isbeyond the range of 5.0 to 8.5. When the pH is high within the aboverange, a strong luminescence unit can be obtained. However, thehalf-life period is shortened. In contrast, when the pH is low, theluminescence unit is small. However, a long half-life period can beobtained. That is, the combination variation of pH and a buffercomponent thereof can adjust luminescence unit and the half-life periodof luminescence to some extent when a luminescence solution of the samecomposition is used. The buffer solution component of controlling a pH,may be any one unless it does not prevent a luciferase activity. Thebuffer solution includes general buffer solutions such as tris-sulfate,citric acid and succinic acid, Good's buffer solutions such as HEPES andMES and broad buffer solutions such as GTA. The concentration of thebuffer component may be any concentration unless it can sufficientlymaintain a pH in the predetermined range from the initiation ofluminescence to a time when the luminescence is reduced by half.

The luciferase assay reagent of the present invention is prepared so asto initiate a reaction at mixing it with a sample containing luciferaseand to have a concentration of a component concerning a luminescencereaction, such as a luminescence substrate, which concentration issufficient for emitting a lucifecase to be measured. The luminescencereagent includes a kit of a frozen product and a kit of a freeze-driedproduct.

The frozen product is obtained by freezing a component containingluciferin, ATP, CoA, a buffer solution component, magnesium ions, etc.Carbon dioxide is forcedly dissolved into a solution before thefreezing. The frozen product is stored at about -70° C. After the frozenproduct thaws out and reaches room temperature fully, it is used.

The kit of the freeze-dried product comprises a first component obtainedby freeze-drying a solution containing luciferin, ATP and CoA and asecond component of a solution containing a buffer solution componentand magnesium ions. The first component and the second component aremixed right before use. In this case, carbon dioxide is forcedlydissolved into the solution of the second component and stored withsealed. The freeze-dried product and the solution component arefreeze-stored. After the freeze-dried product and the solution componentthaw out and reach room temperature, these components are mixed beforeuse.

Each of the frozen product and the freeze-dried product can be stored ina solution state for 24 hours at room temperature or for a month at lowtemperatures (4° C.) with almost no loss of luminescence properties.

The present invention will be explained more in detail with reference toExamples.

EXAMPLE 1

To 10 ml of a luciferse assay regent of "Pica Gene Luminescence Kit"(product No. "PGL100", supplied by TOYO INK MFG. Co., LTD), a GTA buffersolution whose pH was adjusted at 6.7 was added so as to have an endconcentration of 100 mM. Then, a dry ice was added in an amount of 1 gper 10 ml of the luminescence reagent and the dry ice was completelyvaporized. As a stabilizer for luciferase, glycerol was added so as tohave an end concentration of 1%, and the mixture was mixed well toprepare a luminescence solution.

10 μl of an enzyme solution containing 100 ng/ml of luciferase was takenonto a measurement cuvette for a luminometer ("LB9506", supplied byBerthold). Then, 100 μl of the above prepared luminescence solution wasadded, and immediately, the cuvette was set on the luminometer.Luminescence units with the passage of time were measured from rightafter the addition of the reagent (0 minute) to after seven hours (420minutes). In the luminescence reaction by the above luminescencereagent, the luminescence unit (RLU/sec) was stabilized at anapproximately constant value after 5 minutes from the initiation, thoughthe luminescence unit was in the increasing direction. 30 minutes afterthe initiation, the luminescence unit reached its peak and, then, it wasgradually attenuated. Thereafter, two hours after the initiation, theluminescence unit came to 50% of the maximum luminescence unit, to reacha half-life period.

Further, when the luciferase enzyme solution was diluted and the dilutedsolution in the range of 10 ng (10⁻⁹ g) to 1 fg (10⁻¹⁵ g) was added, 10fg of luciferase enzyme was detected the above luminescence solution.

Further, the prepared luminescence solution was frozen and defrostedthree times. Almost no reduction of a luminescence activity wasobserved. The prepared luminescence solution after 1-month storage at 4°C. showed no activity reduction.

EXAMPLE 2

To 10 ml of a luciferase assay regent of "Pica Gene Luminescence Kit"(product No. "PGL100", supplied by TOYO INK MFG. Co., LTD), a citricacid buffer solution whose pH was adjusted at 6.3 was added so as tohave an end concentration of 100 mM. Then, a dry ice was added in anamount of 1 g per 10 ml of the luminescence reagent and the dry ice wascompletely vaporized. As a stabilizer for luciferase, glycerol was addedso as to have an end concentration of 1%, and the mixture was mixed wellto prepare a luminescence solution.

10 μl of an enzyme solution containing 100 ng/ml of luciferase was takenonto a measurement cuvette for a luminometer ("LB9506", supplied byBerthold). Then, 100 μl of the above prepared luminescence solution wasadded, and immediately, the cuvette was set on the luminometer.Luminescence units with the passage of time were measured from rightafter the addition of the reagent (0-minute) to after seven hours (420minutes). In the luminescence reaction by the above luminescencereagent, the luminescence unit (RLU/sec) was stabilized at anapproximately constant value after 10 minutes from the initiation. 60minutes after the initiation, the luminescence unit reached its peakand, then, it was gradually attenuated. Even after 7 hours, theluminescence unit was maintained at 67% of the maximum luminescenceunit.

Further, when the luciferase enzyme solution was diluted and the dilutedsolution in the range of 10 ng to 1 fg was added, 100 fg of luciferaseenzyme was detected with the above luminescence solution.

Further, the prepared luminescence solution was frozen and defrostedthree times. Almost no reduction of a luminescence activity wasobserved. The prepared luminescence solution after 1-month storage at 4°C. showed no activity reduction.

EXAMPLE 3

To 10 ml of a luciferse assay regent of "Pica Gene Luminescence Kit"(product No. "PGL100", supplied by TOYO INK MFG. Co., LTD), a GTA buffersolution whose pH was adjusted at 6.7 was added so as to have an endconcentration of 100 mM. Then, a carbon dioxide gas was introduced intothe reagent solution with a gas introducing tube at a flow rate of 20ml/min for about 3 minutes while bubbling lightly. As a stabilizer forluciferase, glycerol was added so as to have an end concentration of 1%,and the mixture was mixed well to prepare a luminescence solution.

10 μl of an enzyme solution containing 100 ng/ml of luciferase was takenonto a measurement cuvette for a luminometer ("LB9506", supplied byBerthold). Then, 100 μl of the above prepared luminescence solution wasadded, and immediately, the cuvette was set on the luminometer.Luminescence units with the passage of time were measured from rightafter the addition of the reagent (0 minute) to after seven hours (420minutes). In the luminescence reaction by the above luminescencereagent, the luminescence unit (RLU/sec) was stabilized at anapproximately constant value after 5 minutes from the initiation, thoughthe luminescence unit was in the increasing direction. 30 minutes afterthe initiation, the luminescence unit reached its peak and, then, it wasgradually attenuated. Thereafter, two hours after the initiation, theluminescence unit came to 50% of the maximum luminescence unit, to reacha half-life period.

Further, when the luciferase enzyme solution was diluted and the dilutedsolution in the range of 10 ng to 1 fg was added, 10 fg of luciferaseenzyme was detected with the above luminescence solution.

Further, the prepared luminescence solution was frozen and defrostedthree times. Almost no reduction of a luminescence activity wasobserved. The prepared luminescence solution after 1-month storage at 4°C. showed no activity reduction.

Comparative Example 1

10 μl of an enzyme solution containing 100 ng/ml of luciferase was takenonto a measurement cuvette for a luminometer ("LB9506", supplied byBerthold). "Pica Gene Luminescence Kit" (product No. "PGL100", suppliedby TOYO INK MFG. Co., LTD) was prepared as a conventional luminescencereagent. 100 μm of the prepared reagent is added to the cuvette.Immediately, the cuvette was set on the luminometer. Luminescence unitswith the passage of time were measured from right after the addition ofthe reagent (0-minute) to after seven hours (420 minutes). From theinitiation of the luminescence reaction, very high luminescence unitswere obtained. However, the luminescence unit reached a half-life periodafter about 5 minutes. 2 hours after the initiation of the reaction, theluminescence unit was reduced to 1.4% of the maximum luminescence unit(RLU/sec). Further, 7 hours after the initiation, it was reduced to 0.7%of the maximum luminescence unit (RLU/sec) . When the preparedluminescence solution was frozen and defrosted three times, theluminescence activity was reduced to 90%. The luminescence solutionafter 1-month storage at 4° C. was reduced in activity to 10% or less.

Table 1 shows the measured results in Examples and Comparative Example.The method of the present invention shows superior results incontinuation and stability of luminescence to those of the conventionalmethod.

                  TABLE 1                                                         ______________________________________                                                 Variations of luminescence unit with the                               passage of time and retaining ratios of                                       luminescence unit                                                           Hours passed                                                                             CEx.1     Ex.1     Ex.2   Ex.3                                     ______________________________________                                          0 hour   2,270,882 698,851  44,725 701,274                                     100% 100% 100% 100%                                                          0.5 hour 689,060 716,254 42,541 712,527                                        30.3% 102.5% 95.1% 101.6%                                                      1 hour 149,067 581,821 42,839 582,496                                        6.6% 83.3% 95.8% 83.1%                                                         2 hours 31,283 357,833 40,710 358,285                                        1.4% 51.2% 91.0% 51.1%                                                         3 hours 17,178 200,816 38,503 201,224                                        0.8% 28.7% 86.1% 28.7%                                                         5 hours 7,433 79,764 33,771 80,012                                           0.3% 11.4% 75.5% 11.4%                                                         8 hours 3,935 38,641 28,940 38,851                                           0.2% 5.5% 64.7% 5.5%                                                       ______________________________________                                         Note; 100 μl of each luminescence reagent was added to 1 ng/10 μl o     a luciferase solution, luminescence units with time from right after the      reaction initiation were measured with a luminometer ("LB9506", supplied      by Berthold), to measure a relative luminescence unit per unit second         (RLU/s).                                                                 

UTILITIES IN INDUSTRY

When the luminescence solution prepared by the present invention isused, stability in luminescence is remarkably increased as compared withthe conventional one having a half-life period of luminescence of 5minutes. When the luminescence solution having a high pH is used, thehalf-life period is increased by 20 times or more at a detectionsensitivity of 10 fg. When the luminescence solution having a low pH isused, the half-life period is increased by 90 times or more at adetection sensitivity of 100 fg.

As described above, according to the present invention, there isprovided a luminescence measuring method excellent in continuation andstability instead of the conventional luminescence measuring method ofluciferase, which method is featured by rapidity and high sensitivity.

What is claimed is:
 1. A luminescence method for luciferin/luciferasesystem, which comprises reacting luciferin solution containing anincreased amount of dissolved carbon dioxide with luciferase.
 2. Theluminescence method according to claim 1, wherein the pH of theluciferin solution is adjusted in the range of from 5.0 to 8.5.
 3. Theluminescence method according to claim 1, wherein the amount of thedissolved carbon dioxide in the luciferin solution is increased byadding a dry ice.
 4. The luminescence method according to claim 1,wherein the amount of the dissolved carbon dioxide in the luciferinsolution is increased by introducing a carbon dioxide gas.
 5. Aluciferase assay reagent which comprises a luciferin solution containingan increased amount of dissolved carbon dioxide.
 6. A luciferase assayreagent which comprises a first component containing a dried luciferinand a second component of a solution containing an increased amount ofdissolved carbon dioxide.